DNA ligases are ubiquitous enzymes that catalyze an essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. RNA ligases participate in breakage-repair pathways that underlie tRNA splicing, post-transcriptional RNA editing, and cellular stress responses. DNA and RNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-N-zeta)-AMP intermediate; (ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA or AppRNA); (iii) ligase directs an attack by the 3'-OH on AppDNA/RNA to form a phosphodiester bond and release AMP. Our aims are to understand how ligase reaction chemistry is catalyzed and how ligases recognize damaged DNA or RNA ends. We study these problems using three model systems: a eukaryal virus-encoded DNA ligase (Chlorella virus DNA ligase: ChVLig); a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a viral ATP-dependent RNA ligase (T4 Rnl2). Many physiologically important types of DNA and RNA damage result in strand breaks with 3'-PO4 or 2',3'-cyclic-PO4 (>p) ends, which cannot be sealed by classic DNA/RNA ligases. Such broken ends must be healed - converted to a 3'-OH by a phosphoesterase - before they can be sealed. Nature has devised a remarkably diverse enzymatic tool-kit to deal with the end-healing problem. We are focused in this project on two types of end-healing systems: T4 polynucleotide kinase-phosphatase (Pnkp) and LigD phosphoesterase (LigD PE). T4 Pnkp exemplifies a large family of DNA and RNA repair proteins that convert 3'-PO4/5'-OH (or 2',3'>p/5'-OH) ends into ligatable 3'-OH/5'-PO4 ends. The mechanism of RNA cyclic-phosphate removal by T4 Pnkp is unique and entails four chemical steps and two covalent enzyme- substrate intermediates. LigD PE catalyzes two types of end-healing reactions on a DNA primer-template containing either a 3'-diribonucleotide or a 3'-PO4. The 3'-terminal nucleoside of a 3'-diribonucleotide is removed by LigD PE's phosphodiesterase activity to yield a primer strand with a ribonucleoside 3'-PO4. The 3'-PO4 is hydrolyzed by a LigD PE phosphomonoesterase activity to a 3'-OH. The atomic structure of the LigD PE domain and its active site are novel. Indeed, LigD PE defines a new superfamily of repair enzymes distributed widely in bacteria, archaea, and eukarya. We aim to understand how T4 Pnkp and LigD PE enzymes recognize their substrates and cofactors and perform their distinctive phosphoryl transfer chemistries. We propose a multidisciplinary agenda, blending biochemistry, molecular genetics, and structural biology. Our experiments will yield new insights to phosphoryl transfer reaction mechanisms and the evolution of nucleic acid repair systems. .